Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
In recent years, non-antigenic water-soluble polymers, such as polyethylene glycol ("PEG"), have been used for the covalent modification of polypeptides of therapeutic and diagnostic importance. For example, covalent attachment of PEG to therapeutic polypeptides such as interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15,064; Tsutsumi, Y. et al., J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al., Drug Des. Delivery 1990, 6, 157), catalase (Abuchowski, A. et al., J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al., Anal. Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al., Biochim. Biophy. Acta 1981, 660, 293), has been reported to extend their half life in vivo, and/or reduce their immunogenicity and antigenicity.
However, such methods have serious drawbacks. Specifically, in most instances, PEG molecules are attached through amino groups on polypeptides using methoxylated PEG ("mPEG") having different reactive moieties. Such polymers include mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate, mPEG-imidate, and mPEG-cyanuric chloride. The attachment using these polymers was usually non-specific, i.e., occurring at various amino groups on the polypeptides in a random fashion, and not exclusively at a particular amino group. Such non-specific attachment may modify amino acid residues at active sites in such a manner as to eliminate the biological activity of the polypeptides. Also, the resultant conjugates may contain a heterogeneous mixture of modified polypeptide, which is undesirable for pharmaceutical use.
To overcome these problems, it was desirable to site-specifically attach a polymer to a polypeptide. For the polypeptide, doing so would preserve biological activity, prolong blood circulating time, reduce immunogenicity, increase aqueous solubility, and enhance resistance to protease digestion. Site-specific pegylation at the N-terminus, side chain and C-terminus of a potent analog of growth hormone-releasing factor has been performed through solid-phase synthesis(Felix, A. M. et al., Int. J. Peptide Protein Res. 1995, 46, 253). Since the specific pegylation was accomplished during assembly of the peptide on a resin, the method can not be applied to an existing peptide.
An additional method used involved attaching a peptide to extremities of liposomal surface-grafted PEG chains in a site-specific manner through a reactive aldehyde group at the N-terminus generated by sodium periodate oxidation of N-terminal threonine (Zalipsky, S. et al., Bioconj. Chem. 1995, 6, 705). However, this method is limited to polypeptides with N-terminal serine or threonine residues.
Enzyme-assisted methods for introducing activated groups specifically at the C-terminus of a polypeptide have also been described (Schwarz, A. et al., Methods Enzymol. 1990, 184, 160; Rose, K. et al., Bioconjugate Chem. 1991, 2, 154; Gaertner, H. F. et al., J. Biol. Chem. 1994, 269, 7224). Typically, these active groups can be hydrazide, aldehyde, and aromatic-amino groups for subsequent attachment of functional probes to polypeptides. However, since the methods are based on the specificity of proteases, they require extreme caution, and the scope of their application is limited.
Site-specific mutagenesis is a further approach which has been used to prepare polypeptides for site-specific polymer attachment. WO 90/12874 describes the site-directed pegylation of proteins modified by the insertion of cysteine residues or the substitution of other residues for cysteine residues. This publication also describes the preparation of mPEG-erythropoietin ("mPEG-EPO") by reacting a cysteine-specific mPEG derivative with a recombinantly introduced cysteine residue on EPO. Similarly, interleukin-2 was pegylated at its glycosylation site after site-directed mutagenesis (Goodson, R. J. et al., Bio/Technology 1990, 8, 343).
Glycoproteins provide carbohydrates as additional target sites for modification. The enzyme peroxidase has been modified with PEG-diamine through its carbohydrate moiety (Urrutiogoity, M. et al., Biocatalysis 1989, 2, 145). WO 94/28024 describes the methods for preparing mPEG-EPO through periodate-oxidized carbohydrate. The chemistry involved was hydrazone formation by reacting mPEG-hydrazide with aldehyde groups of the carbohydrate moiety on EPO. This type of modification generates reactive aldehyde groups through an oxidation step, which potentially can oxidize various types of sugar residues in the carbohydrate moiety and some amino acid residues in the polypeptide, such as methionine. Another disadvantage of this method stems from the heterogeneity of the carbohydrate moieties of EPO. EPO expressed from Chinese hamster ovary cells has four carbohydrate chains, which include three N-linked chains at asparagines 24, 38, and 83 and one O-linked chain at serine 126. A total of 52 different N-linked, and at least 6 O-linked, oligosaccharide structures have been identified (Rush, R. S. et al., Anal. Chem. 1995, 67, 1442; Linsley, K. B. et al., Anal. Biochem. 1994, 219, 207). Accordingly, it is difficult to control the number of, or attachment sites of, polymer molecules when modifying EPO or other protein via its carbohydrate chains.
In short, the methods in the art for attaching a water-soluble polymer to a polypeptide suffer from serious drawbacks. These drawbacks include the following: (a) a lack of precision, both stoichiometrically and with respect to the situs of attachment; (b) the need to perform difficult and labor-intensive techniques such as site-specific mutagenesis; (c) the need to use solid-phase peptide synthesis concurrently with polymer attachment, instead of attaching a polymer to a pre-existing polypeptide; and (d) the rigid requirement that the identity of the N-terminal amino acid residue be threonine or serine.
For some time, there has existed a need for a general method of site-specifically attaching a water-soluble polymer to the N-terminal amino acid residue of a polypeptide, which method does not suffer from the above-identified drawbacks. However, no such method exists.